Cemtirestat, an aldose reductase inhibitor and antioxidant compound, induces ocular defense against oxidative and inflammatory stress in rat models for glycotoxicity.

Fructose, endogenously produced as a consequence of activation of the polyol pathway under hyperglycemic conditions, contribute to formation of advanced glycoxidation end products (AGEs) and carbonyl stress. Oxidative stress is increased in diabetes (DM) due to AGEs formation and the utilization of NADPH by aldo-keto reductase, AKR1B1(AR), the first enzyme in polyol pathway. Since inhibition of AR is an attractive approach for the management of diabetic eye diseases, we aimed to compare the effects of a novel AR inhibitor (ARI)/antioxidant (AO) compound cemtirestat on eye tissues with the effects of ARI drug epalrestat and AO agent stobadine in rat model for glycotoxicity. One group of rats was fed high fructose (10% drinking water; 14 weeks), while type-2 DM was induced in the other group of rats with fructose plus streptozotocin (40 mg/kg-bw/day). Diabetic (D) and nondiabetic fructose-fed rats (F) were either untreated or treated with two different doses of cemtirestat (2.5 and 7.5 mg/kg-bw/day), epalrestat (25 and 50 mg/kg-bw/day), or stobadine (25 and 50 mg/kg-bw/day) for 14 weeks. Cemtirestat, epalrestat, and stobadine elaviate the increase in TNF-α, IL-1ß, NF-ƙB, and caspase-3 in retina, lens, cornea, and sclera of F and D rats. Both glycotoxicity models resulted in a decrease in GSH to GSSG ratio and a change in glutathione S-transferase activity in eye tissues, but these alterations were improved especially with cemtirestat and stobadine. Lens D-sorbitol of D rats increased more than that of F rats, this increase was only attenuated by cemtirestat and epalrestat. Epalrestat was more effective than cemtirestat and stobadine in inhibiting the increase of vascular endothelial growth factor (VEGF) in the retina of F and D rats. Cemtirestat and stobadine but not epalrestat decreased high level of Nε-(carboxymethyl)lysine in the lens and retina of F and D rats. Cemtirestat is a potential therapeutic in protecting the rat eye against glycotoxicity insults.

A dual-acting aldose reductase inhibitor impedes oxidative and carbonyl stress in tissues of fructose- and streptozotocin-induced rats: comparison with antioxidant stobadine.

Inhibiting aldose reductase (ALR2, AR) as well as maintaining a concomitant antioxidant (AO) activity via dual-acting agents may be a rational approach to prevent cellular glucotoxicity and at least delay the progression of diabetes mellitus (DM). This study was aimed at evaluating the dual-acting AR inhibitor (ARI) cemtirestat (CMTI) on tissue oxidative stress (OS) and carbonyl stress (CS) biomarkers in rats exposed to fructose alone (F) or fructose plus streptozotocin (D; type-2 diabetic). D and F rats were either untreated or treated daily with low- or high-dose CMTI, ARI drug epalrestat (EPA) or antioxidant stobadine (STB) for 14 weeks. Malondialdehyde (MDA), glutathione S-transferase (GST), nitric oxide synthase (NOS), and catalase (CAT) were increased in the sciatic nerve of F and D. These increases were attenuated by low doses of CMTI and STB in D, but exacerbated by low-dose EPA and high-dose CMTI in F. STB and CMTI and to a lesser extent EPA improved MDA, protein-carbonyl, GST and CAT in the hearts and lungs of F and D. CMTI and STB were more effective than EPA in improving the increased MDA and protein-carbonyl levels in the kidneys of F and especially D. CMTI ameliorated renal GST inhibition in D. In the lungs, hearts, and kidneys of F and D, the GSH to GSSG ratio decreased and caspase-3 activity increased, but partially resolved with treatments. In conclusion, CMTI with ARI/AO activity may be advantageous in overcoming OS, CS, and their undesirable consequences, with low dose efficacy and limited toxicity, compared to ARI or antioxidant alone.

Novel substituted N-benzyl(oxotriazinoindole) inhibitors of aldose reductase exploiting ALR2 unoccupied interactive pocket.

Recently we have developed novel oxotriazinoindole inhibitors (OTIs) of aldose reductase (ALR2), characterized by high efficacy and selectivity. Herein we describe novel OTI derivatives design of which is based on implementation of additional intermolecular interactions within an unoccupied pocket of the ALR2 enzyme. Four novel derivatives, OTI-(7-10), of the previously developed N-benzyl(oxotriazinoindole) inhibitor OTI-6 were synthetized and screened. All of them revealed 2 to 6 times higher ALR2 inhibitory efficacy when compared to their non-substituted lead compound OTI-6. Moreover, the most efficient ALR2 inhibitor OTI-7 (IC50 = 76 nM) possesses remarkably high inhibition selectivity (SF ≥ 1300) in relation to structurally related aldehyde reductase (ALR1). Derivatives OTI-(8-10) bearing the substituents -CONH2, -COOH and -CH2OH, possess 2-3 times lower inhibitory efficacy compared to OTI-7, but better than the reference inhibitor OTI-6. Desolvation penalty is suggested as a possible factor responsible for the drop in ALR2 inhibitory efficacy observed for derivatives OTI-(8-10) in comparison to OTI-7.

Development of Novel Indole-Based Bifunctional Aldose Reductase Inhibitors/Antioxidants as Promising Drugs for the Treatment of Diabetic Complications.

Aldose reductase (AR, ALR2), the first enzyme of the polyol pathway, is implicated in the pathophysiology of diabetic complications. Aldose reductase inhibitors (ARIs) thus present a promising therapeutic approach to treat a wide array of diabetic complications. Moreover, a therapeutic potential of ARIs in the treatment of chronic inflammation-related pathologies and several genetic metabolic disorders has been recently indicated. Substituted indoles are an interesting group of compounds with a plethora of biological activities. This article reviews a series of indole-based bifunctional aldose reductase inhibitors/antioxidants (ARIs/AOs) developed during recent years. Experimental results obtained in in vitro, ex vivo, and in vivo models of diabetic complications are presented. Structure-activity relationships with respect to carboxymethyl pharmacophore regioisomerization and core scaffold modification are discussed along with the criteria of 'drug-likeness". Novel promising structures of putative multifunctional ARIs/AOs are designed.

Non-acidic bifunctional benzothiazole-based thiazolidinones with antimicrobial and aldose reductase inhibitory activity as a promising therapeutic strategy for sepsis.

Sepsis is a life-threatening disease that affects millions of people worldwide. Microbial infections that lead to sepsis syndrome are associated with an increased production of inflammatory molecules. Aldose reductase has recently emerged as a molecular target that is involved in various inflammatory diseases, including sepsis. Herein, a series of previously synthesized benzothiazole-based thiazolidinones that exhibited strong antibacterial and antifungal activities has been evaluated for inhibition efficacy against aldose reductase and selectivity toward aldehyde reductase under in vitro conditions. The most promising inhibitor 5 was characterized with IC50 value of 3.99 µM and a moderate selectivity. Molecular docking simulations revealed the binding mode of compounds at the active site of human aldose reductase. Moreover, owning to the absence of an acidic pharmacophore, good membrane permeation of the novel aldose reductase inhibitors was predicted. Excellent "drug-likeness" was assessed for most of the compounds by applying the criteria of Lipinski's "rule of five".

Design synthesis and evaluation of novel aldose reductase inhibitors: The case of indolyl-sulfonyl-phenols.

Therapeutic interventions with aldose reductase inhibitors appear to be a promising approach to major pathological conditions (i.e. neuropathy/angiopathy related to chronic hyperglycemia, chronic inflammation and cancer). Until now, the most potent aldose reductase inhibitors have been carboxylic acid derivatives, which poorly permeate biological membranes. In this work, continuing our previous works, we promote the bioisosteric replacement of the carboxylic acid moiety to make equally potent yet more druggable inhibitors.

3-Mercapto-5H-1,2,4-Triazino[5,6-b]Indole-5-Acetic Acid (Cemtirestat) Alleviates Symptoms of Peripheral Diabetic Neuropathy in Zucker Diabetic Fatty (ZDF) Rats: A Role of Aldose Reductase.

Peripheral neuropathy is the most prevalent chronic complication of diabetes mellitus. Good glycemic control can delay the appearance of neuropathic symptoms in diabetic patients but it is not sufficient to prevent or cure the disease. Therefore therapeutic approaches should focus on attenuation of pathogenetic mechanisms responsible for the nerve injury. Considering the role of polyol pathway in the etiology of diabetic neuropathy, we evaluated the effect of a novel efficient and selective aldose reductase inhibitor, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (cemtirestat), on symptoms of diabetic peripheral neuropathy in Zucker Diabetic Fatty (ZDF) rats. Since the age of 5 months, male ZDF rats were orally administered cemtirestat, 2.5 and 7.5 mg/kg/day, for two following months. Thermal hypoalgesia was evaluated by tail flick and hot plate tests. Tactile allodynia was determined by a von Frey flexible filament test. Two-month treatment of ZDF rats with cemtirestat (i) did not affect physical and glycemic status of the animals; (ii) partially inhibited sorbitol accumulation in red blood cells and the sciatic nerve; (iii) markedly decreased plasma levels of thiobarbituric acid reactive substances; (iv) normalized symptoms of peripheral neuropathy with high significance. The presented findings indicate that inhibition of aldose reductase by cemtirestat is not solely responsible for the recorded improvement of the behavioral responses. In future studies, potential effects of cemtirestat on consequences of diabetes that are not exclusively dependent on glucose metabolism via polyol pathway should be taken into consideration.

Artichoke leaf extract, as AKR1B1 inhibitor, decreases sorbitol level in the rat eye lenses under high glucose conditions ex vivo.

In the previous study, the artichoke leaf extract showed effective inhibition of AKR1B1, the first enzyme of polyol pathway, which reduces high level of glucose to osmotically active sorbitol, important for development of chronic diabetic complications. In the present study, the effect of artichoke leaf extract and of several participating phenols (caffeic acid, chlorogenic acid, quinic acid, and luteolin) was tested on sorbitol level in rat lenses exposed to high glucose ex vivo, on cytotoxicity as well as on oxidative stress in C2C12 muscle cell line induced by high glucose in vitro. The concentration of sorbitol was determined by enzymatic analysis, the cytotoxicity was provided by WST-1 test and intracellular content of reactive oxygen species was determined by fluorescence of 2'-7'-dichlorofluorescein probe. The extract and the compounds tested showed significant protection against toxic effects of high concentration of glucose in both models. On balance, the artichoke leaf extract thus represents a prospective preventive agent of development of chronic diabetic complications, probably due to phenols content, concerning preclinical and clinical studies.

Structure optimization of tetrahydropyridoindole-based aldose reductase inhibitors improved their efficacy and selectivity.

In our previous study, tetrahydropyridoindoles carboxymethylated in position 8 were identified as aldose reductase (ALR2) inhibitors with mild efficacy and selectivity yet with significant antioxidant activity. In the present study we proceeded with optimization of the tetrahydropyridoindole scaffold by shifting the carboxymethyl pharmacophore from position 8 to position 5, with the aim to improve the biological activity. Commercial databases were screened for the presence of tetrahydropyridoindoles carboxymethylated in position 5 and an experimental set of eight compounds was created. Mild inhibition characterized by IC50 in micromolar range was recorded for compound 8 with the isopropyl substituent at the piperidine nitrogen (position 2). This alkylated tertiary nitrogen is characterized by a rather high basicity (pKa ∼ 10.4) with complete protonization at physiological pH. On the other hand, ALR2 inhibition activity of the low basicity derivatives 3-7 with an acyl substituted nitrogen in position 2 (pKa ∼ -1 to -3) was characterized with IC50 values in low and medium nanomolar region. Docking into the binding site of human recombinant enzyme AKR1B1 performed for 3 revealed an interaction network responsible for the high affinity and selectivity. In ex vivo experiment, sorbitol accumulation in isolated rat eye lenses was significantly inhibited by 3 in the presence of high glucose, starting at a concentration as low as 0.1 µM. Moreover, in streptozotocin-induced diabetic rats, compound 3 administered intragastrically (i.g., 50 mg/kg/day) for five consecutive days significantly inhibited sorbitol accumulation in red blood cells and the sciatic nerve. Molecular obesity indices predicted along with water solubility point an excellent "lead-likeness" of compound 3, with prospects of further structure optimizations.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

CHNQ, a novel 2-Chloro-1,4-naphthoquinone derivative of quercetin, induces oxidative stress and autophagy both in vitro and in vivo.

Quercetin (Qc) shows strong antitumor effects but has limited clinical application due to poor water solubility and bioavailability. In a screening of novel semi-synthetic derivatives of Qc, 3,7-dihydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxyphenyl]-5-hydroxychromen-4-one (CHNQ) could ameliorate acetic acid induced acute colitis in vivo more efficiently than Qc. Since inflammation contributes to colorectal cancer (CRC), we have hypothesized that CHNQ may have anti-cancer effects. Using CRC cell lines HCT-116 and HT-29, we report that CHNQ was three-fold more cytotoxic than Qc along with a robust induction of apoptosis. As expected from naphthoquinones such as CHNQ, a strong induction of oxidative stress was observed. This was accompanied by reactive oxygen species (ROS) induced autophagy marked by a dramatic increase in the lipidation of LC3, decreased activation of Akt/PKB, acidic vesicle accumulation and puncta formation in HCT-116 cells treated with CHNQ. Interestingly, an incomplete autophagy was observed in HT-29 cells where CHNQ treatment led to LC3 lipidation, but not the formation of acidic vacuoles. CHNQ-induced cytotoxicity, ROS formation and autophagy were also detected in vivo in Saccharomyces cerevisiae strain RDKY3615 (WinstonS288C background). Overall, we propose that CHNQ can induce cancer cell death through the induction of oxidative stress, and may be examined further as a potential chemotherapeutic drug.

2-Chloro-1,4-naphthoquinone derivative of quercetin as an inhibitor of aldose reductase and anti-inflammatory agent.

The ability of flavonoids to affect multiple key pathways of glucose toxicity, as well as to attenuate inflammation has been well documented. In this study, the inhibition of rat lens aldose reductase by 3,7-di-hydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxy-phenyl]-5-hydroxy-chromen-4-one (compound 1), was studied in greater detail in comparison with the parent quercetin (compound 2). The inhibition activity of 1, characterized by IC50 in low micromolar range, surpassed that of 2. Selectivity in relation to the closely related rat kidney aldehyde reductase was evaluated. At organ level in isolated rat lenses incubated in the presence of high glucose, compound 1 significantly inhibited accumulation of sorbitol in a concentration-dependent manner, which indicated that 1 was readily taken up by the eye lens cells and interfered with cytosolic aldose reductase. In addition, compound 1 provided macroscopic protection of colonic mucosa in experimental colitis in rats. At pharmacologically active concentrations, compound 1 and one of its potential metabolite 2-chloro-3-hydroxy-[1,4]-naphthoquinone (compound 3) did not affect osmotic fragility of red blood cells.

Protection or cytotoxicity mediated by a novel quinonoid-polyphenol compound?

Many natural and synthetic quinones and naphthoquinones possess a variety of beneficial pharmacological properties. In plants, the cytotoxic properties of quinones serve in their defensive roles against invading bacteria, fungi and parasites. In this regard many quinones as well as polyphenols, exerting generally toxicity at high dosages, are able to induce favorable hormetic responses at a low dosage. The novel chloronaphthoquinone derivative of quercetin (CHNQ) showed a profound cytotoxicity followed by enhancement of intracellular generation of oxidants in human neonatal B-HNF-3 fibroblasts. Its synthetic precursors, quercetin and 2-chloro-3-hydroxy-[1,4]naphthoquinone, failed to induce these effects, and paradoxically, only CHNQ at a low concentration provided partial protection of the cells against oxidative challenge. Thus, the novel quinonoid-polyphenol CHNQ might have a merit in the search for new prospective agents in prevention and management of ageing and ageing-related pathologies.

Decreasing acidity in a series of aldose reductase inhibitors: 2-Fluoro-4-(1H-pyrrol-1-yl)phenol as a scaffold for improved membrane permeation.

Targeting long-term diabetic complications, as well as inflammatory pathologies, aldose reductase inhibitors (ARIs) have been gaining attention over the years. In the present work, in order to address the poor membrane permeation of previously reported ARIs, derivatives of N-phenylpyrrole, bearing groups with putative pKa≥7.4, were synthesized and evaluated for aldose reductase inhibitory activity. The 2-fluorophenol group proved the most promising moiety, and further modifications were explored. The most active compound (31), identified as a submicromolar inhibitor (IC50=0.443µM), was also selective against the homologous enzyme aldehyde reductase. Cross-docking revealed that 31 displays a peculiar interaction network that may be responsible for high affinity. Physicochemical profiling of 31 showed a pKa of 7.64, rendering it less than 50% ionized in the physiological pH range, with potentially favorable membrane permeation. The latter was supported from the successful inhibition of sorbitol formation in rat lenses and the ability to permeate rat jejunum.

Cemtirestat dimerization in liposomes and erythrocytes exposed to peroxyl radicals was reverted by thiol-disulfide exchange with GSH.

In the model system of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) liposomes exposed to peroxyl radicals generated by the azoinitiator AAPH, cemtirestat (CMTI-SH) inhibited lipid peroxidation more efficiently than the natural antioxidant glutathione. In the concentrations 100 to 500 µM, both CMTI-SH and GSH induced distinct lag phases in the initial stages of lipid peroxidation yet GSH produced consistently shorter induction periods (about twice) than equimolar CMTI-SH. Moreover, concentration dependence of lipid peroxidation inhibition measured at the 80th minute, revealed about three times higher IC50 value for GSH compared to CMTI-SH. When the incubations prolonged till 180 min no further absorbance changes at 270 and 302 nm, respectively, occurred. After addition of the reducing agent tris(2-carboxyethyl)phosphine, the absorbance peak at 270 nm shifted back to 302 nm. These findings pointed to the presence of reducible CMTI-SH disulfide whose definite structure was confirmed by proving identity of TLC retention and spectral data with those of the synthesized CMTI disulfide. When CMTI-SH and GSH were present simultaneously in the liposomal incubations, the mixing effect on the induction period was synergistic rather than additive. This was explained by ability of GSH to reduce CMTI disulfide which was proved in separate experiments with an authentic CMTI disulfide prepared synthetically. This finding was also demonstrated by experiment with CMTI-disulfide to protect the erythrocytes against oxidative damage induced by peroxyl radicals. To conclude, CMTI-SH scavenges reactive oxygen species yielding CMTI disulfide while GSH maintains CMTI-SH in the reduced state. This finding was also demonstrated by experiment with CMTI-disulfide to protect the erythrocytes against oxidative damage induced by peroxyl radicals. CMTI-SH would thus represent the first line of the cellular defense against peroxyl radical mediated oxidative stress.

Cemtirestat inhibited lipid peroxidation more efficiently than GSHCemtirestat disulfide was proved as the main oxidation productCemtirestat disulfide protected erythrocytes against oxidative damageCemtirestat disulfide was readily reduced by GSHMechanism of thiol-disulfide exchange reaction was suggested.

Novel rhodanine based inhibitors of aldose reductase of non-acidic nature with p-hydroxybenzylidene functional group.

Aldose reductase, the first enzyme of the polyol pathway represents a key drug target in therapy of diabetic complications. In this study a series of six novel rhodanine based inhibitors of aldose reductase was designed, synthesized, and tested for their ability to inhibit aldose reductase and for selectivity relative to structurally related aldehyde reductase. Aldose reductase inhibitory activities of the compounds were characterized by the IC50 values ranging from 2000 nM to 20 nM. The values of selectivity factors relative to aldehyde reductase were decreasing in the same array from 24 to 5. In silico docking into the inhibitor binding site of aldose reductase revealed a specific binding pattern of the compounds comprising interaction of the deprotonated 4-hydroxybenzylidene group with the anion-binding sub-pocket of aldose reductase, creating a strong H-bond and charge interactions. Predicted pH-distribution profiles of the novel compounds into octanol, supported by experimentally determined distribution ratios, favour drug uptake at the physiological pH, as a result of the presence of the low-acidic phenolic group, instead of the more acidic carboxymethyl functional group.

The Effects of Prolonged Treatment with Cemtirestat on Bone Parameters Reflecting Bone Quality in Non-Diabetic and Streptozotocin-Induced Diabetic Rats.

Cemtirestat, a bifunctional drug acting as an aldose reductase inhibitor with antioxidant ability, is considered a promising candidate for the treatment of diabetic neuropathy. Our study firstly examined the effects of prolonged cemtirestat treatment on bone parameters reflecting bone quality in non-diabetic rats and rats with streptozotocin (STZ)-induced diabetes. Experimental animals were assigned to four groups: non-diabetic rats, non-diabetic rats treated with cemtirestat, diabetic rats, and diabetic rats treated with cemtirestat. Higher levels of plasma glucose, triglycerides, cholesterol, glycated hemoglobin, magnesium, reduced femoral weight and length, bone mineral density and content, parameters characterizing trabecular bone mass and microarchitecture, cortical microarchitecture and geometry, and bone mechanical properties were determined in STZ-induced diabetic versus non-diabetic rats. Treatment with cemtirestat did not affect all aforementioned parameters in non-diabetic animals, suggesting that this drug is safe. In diabetic rats, cemtirestat supplementation reduced plasma triglyceride levels, increased the Haversian canal area and slightly, but insignificantly, improved bone mineral content. Nevertheless, the insufficient effect of cemtirestat treatment on diabetic bone disease does not support its use in the therapy of this complication of type 1 diabetes mellitus.

Rational Design of Highly Potent SARS-CoV-2 nsp14 Methyltransferase Inhibitors.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

Structure-activity relations on [1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenylmethanone. The effect of methoxy substitution on aldose reductase inhibitory activity and selectivity.

Based on our previous work, we studied the effect of methoxy-substitution as well as the regioposition of the benzoyl-moiety of 4a [(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(phenyl)methanone]. On this basis, compounds 4b-c and 5a-c were synthesized and assayed for aldose and aldehyde reductase inhibitory activity. Furthermore, a 4,6-difluoro-5-hydroxyphenyl pattern (9) was studied, in order to verify the optimum position of the phenol-moiety. Compound 5b emerged as the most potent and selective inhibitor. Moreover, further assays proved 5b as a potent antioxidant and an inhibitor of sorbitol accumulation in isolated rat lenses. Combining the above attributes, 5b could serve as a lead compound targeted at long-term diabetes complications.